1. Field of the Invention
This invention relates to the field of diagnostic microbiology and particularly to diagnostic methods for detecting the presence of, and typing of, a microorganism belonging to a specific genus in a biological sample, and to methods for identifying antigens which are common to taxonomically unrelated genera.
2. Brief Description of the Prior Art
The presence of bacteriocin-like activity in isolates of Neisseria gonorrhoeae has been reported by Flynn and McEnteggart, J. Clin. Path. 25:60-61; (1971). Substances from other organisms have also been reported to exhibit bacteriocin-like activity against N. gonorrhoeae. Volk and Kraus, Brit. J. Vener. Dis. 49:511-512 (1973) reported the in vitro inhibition of N. gonorrhoeae by a substance from N. meningitidis. Geizer, J. Hyg. Epid. 12:241-243 (1968) has reported the inhibition of gonoccocal growth by unidentified substances produced by strains of a number of organisms including Pseudomonas aeruginosa. The bacteriocin-like activity exhibited against many strains of N. gonorrhoeae was attributed to the production of inhibitory levels of free fatty acids and lysophosphatidyethanolamine as reported by Walstad, et al., Infect. Immunity, 10:481-488 (1974).
(a) Origin and Structure of Bacteriocins
Bacteriocins are a group of specific bacteriacidal substances produced by many bacterial during growth. They are proteins of varying molecular weight. Bacteriocins have antibiotic properties, but in contrast to antibiotics which are in clinical use, are much more specific, acting only on members of the same or closely related species. They are extracellular substances which become bound to receptor sites of susceptible organisms. Bacteriocins usually remain contained within the producer strain until released by cell lysis. These extracellular substances can then bind to receptor sites of susceptible organisms. Some bacteriocins closely resemble parts of bacteriophage when examined microscopically. Their production can be induced by agents which interfere with metabolism, such as ultra violet light, mytomicin C, nitrogen mustard and many other agents.
There are two basic types of bacteriocins. One type is a small molecule which is thermo-stable, which cannot be sedimented in the ultra centrifuge, and is not easily resolved by the electron microscope. The other, R-type, is larger and resembles phage tails. This difference in basic types is well illustrated by comparing Colicin V with Colicin 15 (colicins being bacteriocins specific to coliform organisms). Colicin V forms a dialyzable product which has a low molecular weight. Colicin 15 is sedimentable, has a molecular weight of 200,000 and, on electron microscopy, resembles the tail structure of a phage.
Small quantities of bacteriocins are released in normal cultures of organisms, and are presumably released during normal lysis found during degeneration of organisms in culture. Their genetic determinants exist as an extra chromosomal element which replicate in phase with bacterial chromosomes, and therefore persist as long as the strain persists. They are released in quantity by lysis of the bacterial cell whether this occurs by phage infection, the action of bacteriolytic agents, such as metabolic inhibitors, or other factors.
Chemically, all bacteriocins are macromolecular and contain polypeptide, protein, other radicals such as carbohydrate, phosphate and lipopolysaccharide which contributes to the ultimate size of the molecule.
(b) Nomenclature
While classification and nomenclature are necessarily undergoing change as more evidence of their origin, chemistry and activities accumulate, bacteriocins are named, as a general rule, on the specific rather than generic name of the originating organisms. For example, E. coli bacteriocins are termed colicins. Serratia marcescens give marcesins; Enterobacteraerogenes, aerocins; Pseudomonas aeruginosa (pyocyaneus), pyocins; Listeria monocytogenes, monocins; Staphylococcus sp., staphylocins, etc.
This classification began after Gratia in Belgium first reported that filtrates of a particular strain of E. coli inhibited growth of the same species, the inhibiting factor being called a colicin. Some 20 colicins were subsequently recognized and classified as A-V. Each colicin was specific for a small group of strains of Enterobacteraciae. Each bacteriocin whether from E. coli or other species appears to be specific in action to the same, or to taxonomically related, species of organisms.
(c) Assay of Bacteriocins
The concentration of bacteriocin in a filtrate titrated by placing a drop (10-20 .mu.l) on a lawn culture inoculated witn indicator bacteria (10.sup.7 /ml) of freshly grown cells. After incubation for 18-24 hours at 37.degree. C the plates are read and scored. Titers are regarded as a reciprocal of the highest dilution that yields a clear spot. Another method is to add bacteriocin to an enumerated excess of sensitive organisms, the bacteriacidal activity being proportional to the quantity of bacteriocin present.
One unit of bacteriocin activity is the lowest concentration which completely inhibits growth of an indicator strain.
At the present time purification is more a matter of concentration from the original broth or saline suspensions then isolation of specific fractions. The most commonly used method to remove the cells by centrifugation after 6-24 hours of incubation. Purification is completed by column chromatography following ammonium sulfate precipitation followed by dialysis against equilibration buffer.
Purified bacteriocins are stable in a lyophilized state for long periods of time. In solution they are stable at 4.degree. C and pH 7.0 for 6 weeks. Bacteriocin are not irreversibly denatured by 4M guanidine thiocyanate or 6M urea, but are completely inactivated at 60.degree. C and pH 7.0 in 60 minutes.
(e) Mode of Action
Bacteriocins act on cells which are in the logarithmic phase of proliferation. The treatment of sensitive cells rapidly inhibits incorporation of labeled leucine and thymidine into acid insoluble materials. The time required for inhibition of .sup.14 C and .sup.3 H labeled isotopes is dependent upon the concentration of bacteriocins. It has been established that both DNA and protein synthesis are blocked by bacteriocin. When organisms in cultures are exposed to bacteriocin, they do not incorporate .sup.14 C leucine into the protein or .sup.3 H thymidine into DNA. This is probably due to interference with the active transport of leucine and thymidine by the specific bacteriocin. Concurrently the concentration of ATP falls to 10-15% of control value. This is not related to a decline in macromolecular synthesis, but does help to explain the faltering energy transport mechanisms which are seen in bacterial cells exposed to bacteriocins. Though ATP activity is inhibited, the phosphotransferase system is not affected and a-methyl D-glucoside has been shown to accumulate in coliforms. Apart from these modes of activity, interference with cell membrane integrity may occur in some species of susceptible organisms.